Use of chemical sensor

ABSTRACT

A chemical sensor ( 1 ) for selectively detecting an analyte in a solution as described. The sensor comprises a flow-through chamber ( 2 ), a selective membrane ( 3 ), a transducer means ( 4 ), an inlet ( 5 ) for a liquid flow containing a recognition element, and an outlet ( 6 ). There is also described a method of selectively detecting an analyte in a solution, wherein a recognition element is contacted with the solution containing the analyte via a selective membrane, said contact resulting in a response detectable by transducer means. The recognition element is injected into a flow, the flow is passed into a flow-through chamber comprising a transducer means and the selective membrane, where it is contacted with the analyte passing from the solution outside the selective membrane, whereby the recognition element and the analyte interact to provide a signal which is detected by the transducer means. The use of the chemical sensor for detecting of analyte(s) in a reactor system, a flow system or in an in vivo system is also described.

[0001] The present invention comprises a chemical sensor for selectivelydetecting an analyte in a solution, a method of selectively detecting ananalyte in a solution, and use of the chemical sensor.

[0002] A chemical sensor is a device which selectively detects a targetmolecule (analyte) in a complex medium (the sample solution) andprovides an output signal which is proportional to the concentration ofthe studied analyte. A chemical sensor consists of two neighbouringcomponents, the so-called recognition element and the transducercomponent. The function of the recognition element is to selectivelybind to the analyte located in the sample solution. In binding, this orsubsequent chemical events should be converted into a quantifiableelectric output signal of the transducer component. A large number ofcombinations of different recognition elements and transducer componentshave previously been reported 1, 2, 3. The classification of therecognition element can be effected on the basis of a biologic ornon-biologic origin, and if it has catalytic or non-catalytic properties(see Table 1). The transducer component is based on different operatingprinciples which can be of electrochemical, optical, magnetic,acoustic/piezo-electric or thermometric nature.

[0003] The capacity of a chemical sensor can be described by parameters,such as selectivity, sensibility, stability, response time andre-usability. A large number of different sensor concepts have beenpresented, of which the so-called biosensors have shown very promisingproperties in respect of selectivity and sensibility to a large numberof analytes. Unfortunately, the stability is not good owing to theirfunction being based on recognition elements of biological origin.

[0004] Enfors, S-O and Nilsson, H, 1979 (4) described a biosensor with amanually exchangeable recognition element. This biosensor is operated byinjecting a solution of an enzyme into the biosensor. The response ismeasured as a pH signal. A large excess of enzyme solution must be used.In order to get a relevant value of the pH response, the authors statethat the solution must be kept still in the biosensor. Slow regenerationis expected because the analyte and the formed enzymatic products haveto diffuse out from the biosensor prior to new measurments can beinitiated. Furthermore, there must be introduced a second transducercomponent in the system, that is a pH electrode measuring the pH of thesurrounding sample solution.

[0005] GBF, Scientific Annual Report 1990, pp 62-63 and 126-127,Biosensors for Pesticides in Water, describes a biosensor based on aliquid flow, into which different substrates are injected. The flow ispassing a selective membrane reactor, in which antibodies areimmobilised. Thus, this biosensor is based on a flowing analyte detectedby a stationary recognition element.

[0006] Various approaches in Flow Injection Analysis (FIA) requiresample pretreatment, have possible contamination of the flow-throughsystem/detector, operation in harsh chemical environments not possible,no on-line or in situ monitoring is possible, samples must be taken(Ruzicka, J. & Hansen, E. T. ‘Flow Injection Analysis’, John Wiley &Sons, N.Y., 1981).

[0007] In one aspect of the present invention, there is provided a newtype of chemical sensor.

[0008] In another aspect of the present invention, there is provided amethod of selectively detecting an analyte in a solution.

[0009] In yet another aspect of the present invention, there is providedthe use of the sensor according to the invention for differentapplications.

[0010] The invention will now be described in more detail with referenceto the accompanying drawings, in which

[0011]FIG. 1 is a schematic view of an embodiment of the chemical sensoraccording to the invention;

[0012]FIG. 2 is a cross-section of an amperometric, transducer, adaptedfor use in a biosensor according to the invention;

[0013]FIG. 3 is a graph showing the pH response of a urea biosensoraccording to the invention as a function of the urea concentration in asample solution;

[0014]FIG. 4 is a graph showing the pH response of the urea biosensoraccording to the invention as a function of the temperature;

[0015]FIG. 5 is a graph showing the glucose concentration in a fermenteras a function of the fermentation time, measured on undiluted samplesusing a biosensor according to the invention;

[0016]FIG. 6 is a graph showing a comparison of results obtained by abiosensor according to the invention (on undiluted samples) and resultsobtained by HPLC analysis (on diluted samples).

[0017] According to the first aspect of the invention, there is provideda chemical sensor 1 for selectively detecting an analyte in a solution,which comprises a flow-through chamber 2, a selective membrane 3,transducer means 4, an inlet 5 for a liquid flow containing arecognition element, and an outlet 6.

[0018] In its second aspect, the invention provides a method ofselectively detecting an analyte in a solution, wherein a recognitionelement is contacted with the solution containing the analyte via aselective membrane, said contact resulting in a response detectable bytransducer means, whereby the recognition element is injected into aflow, the flow is passed into a flow-through chamber comprising thetransducer means and the selective membrane, where it is contacted withthe analyte passing from the solution outside the selective membrane,whereby the recognition element and the analyte interact to provide asignal which is detected by the transducer means.

[0019] In the third aspect of the invention, there is provided the useof the chemical sensor for detecting of analyte(s) in a reactor system,a flow system or in an in vivo system.

[0020] When, in the present invention, the recognition element is ofbiological origin, there is created a biosensor of a totally new type,showing an up to now not seen, but very important stability togetherwith the promising characteristics shown by prior art biosensors, suchas selectivity and sensibility to a great variety of analytes.

[0021] In addition to the above properties, the invention offers thefollowing beneficial new features and advantages compared to knownchemical sensors, due to the invention being based on injection of therecognition element into the biosensor. Thus, the analyses would be morerapid and the times of response shorter, and in addition it will bepossible to control the risks of contamination and deactivation of therecognition element. The reason for this is that the measurement processis dynamic, which allows initial changes to be put in relation to theconcentration of analyte in the sample solution, and that there is noneed for the recognition element to be regenerated. The invention alsoallows sequential detection of several analytes with one and the sametransducer means. It also permits differential measurements with andwithout recognition elements in order to reduce the effect ofinterfering compounds.

[0022] All these new features and advantages will be of great economicimportance and will give opportunities for new applications in theindustrial, medical and research fields.

[0023] The invention can be used for detecting of analyte(s) in areactor system, a flow system or in an in vivo system. It can also beused for on line and/or in situ measurements, and for measurements athigh temperatures and in harsh chemical environments, such as at high orlow pH values, high salt concentrations and/or in the presence ofdenaturating substances. A further use of the invention is fordifferential measurements with and without a recognition element.

[0024] As transducer means, use can be made of an electro-chemical,optical, magnetic, acoustic/piezoelectric or thermometric transducer, ora combination thereof.

[0025] In an especially preferred embodiment, the transducer means is anamperometric transducer. In another preferred embodiment, the transducermeans is a pH transducer.

[0026] Preferred selective membranes for use in the invention aredialysing membranes, ion exchange membranes, gas permeable membranes,analyte selective membranes and group or single analyte selectivehindering membranes.

[0027] The chemical sensor according to the invention can also comprisemeans for pumping the flow into the flow-through chamber and means forinjecting the recognition element into the flow.

[0028] In another preferred embodiment, the chemical sensor is arrangedfor detecting biological analyte(s) using as a recognition element acatalytic or non-catalytic substance of biological origin. Other typesof recognition elements are set forth in the following table: TABLE 1Examples of different types of recognition elements for use in chemicalsensors³. Catalytic Non-catalytic Biological Enzymes, Micro- Proteins(Con origin organisms, Abzymes, A), Antibodies, Organelles, TissueReceptors, DNA Non-biological Artificial enzymes Solid state, and/orIon-exchange and artificial Neutral carrier origin membranes, Oxides,Conducting poly- mers, Molecularly imprinted polymers

[0029] Specific examples of enzymes for use as recognition elements areoxidases, dehydrogenases and hydrolases.

[0030] The measurements can be performed either by passing therecognition element continuously through the flow-through chamber or bykeeping it stationary in the flow-through chamber during the detection.

[0031] A special advantage of the invention is the possibility of simpleand rapid regeneration of the transducer means and/or the selectivemembrane by passing a buffer or washing solution through the chamber foras short a period as a few minutes. Electrochemical transducers can alsobe regenerated using as electro-cleaning procedure, i.e. sweeping theworking electrode potential between extreme values (for instance ±2V).This is due to the fact that the recognition element is injected intothe flow in contrast to being immobilised. Another great advantage ofthis is that very small amounts of recognition elements are required.Thus, only a volume of the solution of e.g. an enzyme, used asrecognition element, in the order of 1-100 μl is necessary.

[0032] The invention relates to a new type of chemical sensors 1, whoseoperating principle is based on the recognition element being injectible(FIG. 1). A buffer solution A is pumped by means of a pump 7, placedeither on the inlet (5) side or on the outlet (6) and thus working in asuction mode, through the injector 8. The recognition element B isinjected into the buffer solution and entrained into the space betweenthe transducer means 4 and a selective membrane 3. The other side of theselective membrane is in contact with the sample solution E. The analytecan pass through this membrane and interact with the recognitionelement, this chemical process being converted by the transducer meansinto an electric output signal F which is proportional to theconcentration of the analyte in the sample solution. The liquid flow isfinally collected in a waste container S.

[0033] The construction also allows a multisensor function by a sequenceof different recognition elements B1, B2, B3 . . . being injectible, asequential detection of various analytes in a sample solution beingeffected. In addition, a mixture of enzymes could be injected where eachenzyme gives a specific response (pH, H₂O₂, coloured products . . . )which can be detected by one of the transducers used. Moreover, othersubstances, such as reagents, mediators, indicators and stabilisers canbe present together with the recognition element between the transducermeans and the selective membrane. A washing solution can periodically beallowed to pass between the transducer means and the selective membranesuch that a quick regeneration thereof can be effected.

[0034] The invention can be used for detection in reactor systems,closed or open containers (such as fermenters) or in flow systems (alsoin in vivo applications) of one or more analytes.

[0035] In a preferred embodiment of the invention, there is used anamperometric transducer for detecting the analyte. An example of such atransducer for use in a bio-sensor according to the invention, designedto measure in a potentiostatic three-electrode system, is shown in FIG.2. The transducer comprises a platinum wire acting as the workingelectrode, a stainless steel auxiliary electrode, and an externalAg/AgCl reference electrode. The sensor also has an internal referenceelectrode, consisting of a 1 mm diameter silver wire, covered withAg/AgCl.

[0036] The following examples are given only as illustrative examples ofembodiments of the invention, and are not to be construed as limitingthe invention, such as it is defined in the accompanying claims.

EXAMPLE 1

[0037] In order to exemplify the invention, a preliminary study has beenmade, in which a urea(bio)sensor has been constructed according to theprinciple described above, using a pH electrode as transducer means. Asappears from FIG. 3, the pH response of the sensor correlates with thecontent of urea in the sample solution. The range of concentration is0-5 mM, which is clinically relevant.

[0038] Furthermore, the effect of potential interfering substances, suchas,glucose, acetone, citric acid and sodium acetate, was investigated,see Table 2. TABLE 2 Substance Concentration (mM) Change in ΔpH(%)Glucose 28 0 Acetone 6.7 0 Citric acid 0.2 0 Sodium acetate 0.6 0 Coppersulphate 1 −38(0) 2 −65(0)

[0039] These did not have an effect on the pH response. It is awell-known fact that copper ions inhibit the catalytic ability of theused recognition element, the enzyme urea, and therefore a reduction ofthe pH response, when such ions were present in the sample solution, wasno surprise. This effect could, however, be eliminated completely if astabiliser reacting with the copper ions (for instance EDTA which formsa strong complex with the copper ions) was present, i.e. injectedtogether with the enzyme.

[0040] Finally, also the pH response of the urea(bio)sensor was analysedin measurements at increased temperatures, see FIG. 4. In thesemeasurements, the sensor showed its superiority to conventionalbiosensors which are quickly deactivated at high temperatures, such as50° C. In this case, only a 25% reduction of the pH response was noted.

EXAMPLE 2

[0041] In the present study, a biosensor with an injectible recognitionelement (glucose oxidase) was used to determine the glucoseconcentration in crude samples from a fermentation of a willowhydrolysate.

MATERIALS AND METHODS

[0042] Yeast: Baker's yeast, Saccharomyces cerevisiae (Jästbolaget,Sweden) was used in the fermentation.

[0043] Preparation of the lignocellulosic hydrolysate: Willow, Salixcaprea, was subjected to steam-pretreatment at 205° C. for 6 min [2].The pretreated material was washed with water and filtered in a Laroxfilter press unit at a pressure of 14 bar. The cellulose fibres werehydrolysed in a stirred tank using Celluclast (20% w/w) and Novozyme (5%w/w) (Novo Nordisk, Denmark) for 90 h at 30° C., pH 4.8.

[0044] Fermentation: The lignocellulosic hydrolysate was supplementedwith 2.5 g.l⁻¹ yeast extract, 0.25 g.l⁻¹ (NH₄)₂PO₄, 0.0025 g.l⁻¹MgSO₄.7H₂O and 0.1 M NaPO₄, and inoculated to a final cell concentrationof 6 g/l. The fermentation was run in a 22 l fermenter (Bioengineering,Switzerland) containing 16 l of medium at 30° C., pH 5.5, and a stirringspeed of 300 rpm.

[0045] Biosensor measurements of glucose: The glucose concentration insamples taken out of the fermenter was measured, using a biosensoraccording to the invention. The mobile phase buffer used was a 0.1 Msodium phosphate buffer (pH 6.0) containing 0.15 M sodium chloride. Theflow rate was set at 0.2 ml/min. The recognition element, glucoseoxidase solution (1 mg/ml in mobile phase buffer) was injected into themobile phase and when it reached the biosensor chamber, the flow wasstopped. A current reading (ER) was recorded after 48 s. In addition, abackground current reading (Br) was recorded, using the same procedurebut injecting mobile phase buffer instead. The biosensor response wascalculated as the difference (Er−Br) in the two current readings. Thebiosensor was calibrated with standard glucose solutions (0-50 g/l inmobile phase buffer). The fermenter samples were centrifuged (5 min at1000 rpm) in order to remove the yeast cells and thus stop theconsumption of glucose. Biosensor measurements were then performed onthe undiluted samples.

[0046] HPLC analyses of glucose: Samples from the fermentation brothwere filtered through 0.2 μm membrane filters (Sartorius, Germany) andanalysed by HPLC (Shimadzu, Japan). Glucose and ethanol were separatedat 45° C. using an Aminex HPX-87H column (Bio-rad, USA) and detectedwith a refractive index detector (Waters Millipore, USA). As the mobilephase 5 mM H₂SO₄ was used at a flow rate of 0.6 ml/min.

[0047]FIG. 5 shows the glucose concentration in the fermenter as afunction of the fermentation time.

[0048]FIG. 6 shows a comparison of results obtained by the biosensoraccording to the invention (on undiluted samples) and results obtainedby HPLC analysis (on diluted samples).

RESULTS AND DISCUSSION

[0049] In contrast to HPLC measurements, the measurements carried out bythe biosensor according to the invention are not influenced byinterfering compounds present in the complex matrix of a lignocellulosichydrolysate and thus the glucose concentration could be monitored inundiluted samples (FIG. 4). The recognition element, in this caseglucose oxidase, is highly specific and responds only to the targetanalyte. A differential measurement method is used, allowingcompensation for background currents which may arise from directoxidation of sample components. No sample pretreatment was needed, themeasurements were carried out in undiluted, unfiltered samples. Due tothe fact that the yeast cells were not removed early enough before themeasurements according to the invention, the glucose concentrations foreach sample were slightly lower using this technique because ofcontinuing fermentation than those obtained by HPLC analysis, where thesamples were immediately filtered, for removal of cells (FIG. 5). Afterthe measurements carried out by the sensor according to the invention,the HPLC analysis was repeated and the results confirmed the observeddecrease in glucose concentration (data not shown). Whereas one HPLCanalysis of an ethanolic fermentation takes at least 30 min for elutionof all compounds present in the fermentation broth, the analysis timeusing the sensor according to the invention does not exceed 5 min, andno time is needed for regeneration of the sensor, which means that theanalysis frequency can be very high. A very broad concentration range iscovered by the sensor, which can detect concentrations down to 2 μMglucose. The sensor is suitable for in situ monitoring of fermentationprocesses. It can be sterilised in situ, and therefore the risk ofcontamination is minimised. Regeneration can be made if necessaryduring, the fermentation by injection of a cleaning agent or by applyinghigh potentials (±2V) to the electrode. No deactivation of thebiological component will occur, as fresh enzyme solution (10 μl) isinjected for every analysis. Automatisation of the analyses is easilyaccomplished by connection of an autoinjector to the electrode. Atpresent, five different enzymes have been evaluated for use asrecognition elements for the detection of urea, glucose, galactose,lactate and L-amino acids. Determination of several broth componentswill be possible by sequential injection of different enzymes.

[0050] To prove the utility of the invention for measurements in harshchemical environments, the glucose content in orange juice, Coca Cola,Coca Cola Light and Pepsi Cola has been measured with excellent results.

1. A chemical sensor (1) for selectively detecting an analyte in asolution, said sensor comprising a selective membrane (3), transducermeans (4), an inlet (5) for a liquid flow, and an outlet (6),characterised in means (8) for injecting a recognition element into saidflow, and a space provided between the transducer means (4) and theselective membrane (3), the transducer means (4) and the membrane (3)forming a flow-through chamber (2), one side of said membrane (3) beingin contact with said solution containing said analyte, the analytepassing through the membrane (3) and interacting with the recognitionelement, said transducer means (4) converting the interaction into asignal (F) proportional to the concentration of the analyte in saidsolution.
 2. A chemical sensor according to claim 1, wherein thetransducer means (4) is an electrochemical, optical, magnetic,acoustic/piezoelectric or thermometric transducer, or a combinationthereof.
 3. A chemical sensor according to claim 1 or 2, wherein thetransducer means (4) is an amperometric transducer.
 4. A chemical sensoraccording to any one of claims 1-2, wherein the transducer element is apH transducer.
 5. A chemical sensor according to any one of claims 1-4,wherein the selective membrane is chosen from the group consisting ofdialysing membrane, ion exchange membrane, gas permeable membrane,analyte selective membrane and group or single analyte selectivehindering membrane.
 6. A chemical sensor according to any one of claims1-5, characterised in that it further comprises means (7) for pumpingthe flow into the flow-through chamber.
 7. A chemical sensor accordingto any one of claims 1-6, characterised in that it is arranged fordetecting analyte(s) using a recognition element (catalytic ornon-catalytic) of biological origin, that is as a biosensor.
 8. A methodof selectively detecting an analyte in a solution, wherein a recognitionelement is contacted with the solution containing the analyte via aselective membrane, said contact resulting in a response detectable bytransducer means, characterised in that the recognition element isinjected into a flow, the flow is passed into a flow-through chambercomprising the transducer means and the selective membrane, where it iscontacted with the analyte passing from the solution out-side theselective membrane, whereby the recognition element and the analyteinteracts to provide a signal which is detected by the transducer means.9. A method according to claim 8, wherein the flow containing therecognition element is passed continuously through the flow-throughchamber during detection.
 10. A method according to claim 8, wherein theflow containing the recognition element is kept stationary in theflow-through chamber during detection.
 11. A method,according to any oneof claims 8-10, wherein a sequence of different recognition elements arepassed through the chamber.
 12. A method according to any one of claims8-10, wherein a mixture of enzymes is passed through the chamber.
 13. Amethod according to any one of claims 8-12, wherein a buffer or washingsolution is passed through the chamber to regenerate the transducermeans and/or the selective membrane.
 14. A method according to any oneof claims 8-12, wherein the transducer means is regenerated byelectro-cleaning.
 15. A method according to any one of claims 8-14,wherein further substances, chosen from the group consisting ofreagents, mediators, indicators and stabilisers, are introduced into thechamber.
 16. A method according to any one of claims 8-15, wherein thedetection by the transducer means is of electrochemical, optical,magnetic, acoustic/piezoelectric, thermometric nature or a combinationthereof.
 17. A method according to any one of claims 8-16, wherein aleakage of the recognition component(s) to the solution containing theanalyte is prevented or reduced by using as a selective membrane adialysing membrane, ion exchange membrane, gas permeable membrane,analyte selective membrane or group or single analyte selectivehindering membrane.
 18. A method according to any one of claims 8-17,wherein the recognition element is of biological origin.
 19. A methodaccording to any one of claims 8-18, wherein the recognition element ischosen among catalytic or non-catalytic elements.
 20. Use of thechemical sensor as defined in any one of claims 1-7 for detecting ofanalyte(s) in a reactor system, a flow system or in an in vivo system.21. Use of the chemical sensor as defined in any one of claims 1-7 foron line and/or in situ measurements.
 22. Use of the chemical sensor asdefined in any one of claims 1-7 for measurements at high temperatures.23. Use of the chemical sensor as defined in any one of claims 1-7 formeasurements in harsh chemical environments.
 24. Use according to claim23 for measurements at high or low pH values, high salt concentrationand/or in the presence of denaturating substances.
 25. Use of thechemical sensor as defined in any one of claims 1-7 for differentialmeasurements with and without a recognition element.